The use of competitive affinity chromatography for the isolation of proteins which promote transcription.
A method is described for isolating proteins which bind preferentially to specific sequences of DNA and is used to enrich preparations in proteins promoting eukaryotic gene transcription. An ion-exchange fraction which promotes the transcription of the ovalbumin gene in in vitro runoff assays was isolated from the oviducts of diethylstilbesterol-stimulated chicks. This fraction was recirculated between two coupled columns, one containing random sequences of prokaryotic DNA and the other a specific cloned DNA fragment from the 5' region of the ovalbumin gene. Recirculation was performed in the presence of a decreasing salt gradient, after which columns were disconnected and separately eluted. The eluate obtained from the column containing cloned DNA showed a preference for binding to DNA fragments derived from the ovalbumin gene when examined in nitrocellulose filter binding assays. This fraction retained the ability of its precursor to promote gene transcription in a concentration-dependent manner. Active preparations were also examined in assays measuring total RNA synthesis from native DNA templates. Although the fraction isolated from the column of specific DNA had no detectable RNA polymerase activity itself, it enhanced the activity of calf thymus RNA polymerase II. The method presented should find general application in the purification of factors which regulate biological processes by binding to specific sequences of DNA.
Sanzo
M A
MA
Department of Cell Biology, Baylor College of Medicine, Houston, TX.
D000818:Animals; D002645:Chickens; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics; D004268:DNA-Binding Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000494:pharmacology; D009691:Nucleic Acid Denaturation; D010047:Ovalbumin / Q000235:genetics; D014157:Transcription Factors / Q000235:genetics / Q000302:isolation & purification / Q000494:pharmacology; D014158:Transcription, Genetic / Q000187:drug effects; D014357:Trypsin
D004268:DNA-Binding Proteins; D014157:Transcription Factors; D010047:Ovalbumin; D004247:DNA; D014357:Trypsin